Gene expression of the human genome in response to physiological and environmental stimuli is dictated by chemical modifications of the DNA and the DNA-packing histones, as well as transcription factors. This highly complex biological system that operates with a large number and different combinations of epigenetic modifications has defied our full investigation of its basic mechanisms with existing tools. A prime example is the biology of stem cell in which a balance between self-renewal and differentiation lies at the heart of how its chromatin structure enacts different transcriptional programs to instruct pluripotent cell behavior. Yamanaka's reprogramming of somatic cells to induced pluripotent stem cells using four transcription factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) highlights the ultimate control of cell fate by gene transcription. However, the questions on how the OSKM factors transform the cell's epigenome and how their own expression are controlled during stem cell self-renewal and differentiation are yet to be answered. The functional effects of epigenetic modifications are realized by the binding of epigenome readers such as the acetyl-lysine binding bromodomain and the methyl-lysine binding chromodomain that are present in transcription and chromatin regulators. Unlike RNA interference or gene knockout that entails the removal of an entire protein along with all its interactions, a small-molecule inhibitor (chemical probe) could remove a single interaction in a multi-domain protein in its endogenous form, thus providing a much finer tool for the temporal perturbation of the complex biological system. In this multidisciplinary research project, we will develop high affinity and selective chemical probes for a selected group of bromodomains and chromodomains that function in stem cell biology, either individually (i.e. single-target probes) or as a group in a biological pathway (i.e. multi-target probes). Our study uses a coherent set of structural/chemical biology and chromatin/stem cell biology methods being developed by our key investigators with multidisciplinary expertise. We will also develop an Epigenome Reader KnowledgeBase to aid target profiling, design, production and validation of new chemical probes. To attain these goals, we will achieve the following three specific aims: (1) Target profiling of epigenome readers based on their epigenetic functions; (2) Probe development using target structure-guided strategy; and (3) Probe validation in a functional context of gene transcriptional regulation in stem cell biology.